PROJECT SUMMARY The clostridial neurotoxins (composed of the tetanus and botulinum neurotoxins) are among the most toxic agents known to humans and cause life-threatening, paralytic disorders. The potential for major public health impact resulting for an intentional release, combined with the paucity of approved vaccines or therapies has led to the classification of BoNTs as Tier 1, Category A Select Agents. Paradoxically, the highly specific action of BoNT types A and B make them excellent pharmaceuticals for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. Despite many recent advances in understanding the structure-function relationship of clostridial neurotoxins, the molecular events by which the neurotoxin heavy chain (HC) is able to transfer (translocate) its enzymatic domain across the membrane bilayer remains poorly defined. In the current application, we will employ single-particle cryo- electron microscopy to determine medium resolution (4-10 ) structures of tetanus neurotoxin (TeNT) interacting with lipid nanodiscs in various states. In aim 1 we will determine the structure of TeNT bound to small ~100 nanodiscs containing the neuronal receptor ganglioside GT1b. The resulting structure will not only expose how the toxin interacts with GT1b within a membrane environment, but also provide new details on the spatial arrangement of the enzymatic and translocation domains. In aim 2, interfacial and insertion competent forms of TeNT will be generated using larger (~170 ) nanodiscs of sufficient bilayer surface area to allow TeNT to transition at low pH from the bound state to the interfacial/inserted TeNT conformations. These snap shots will provide the direct first evidence testing the assumption that transport of the enzymatic domain across the bilayer is mediated by transit through the lumen of the translocon pore. Realizing these goals is crucial for advancing our understanding of the translocation mechanism from both a structural and kinetic standpoint. Determining the initial structural conformations of TeNT as it transitions from a water soluble to membrane inserted protein will be extremely useful in future efforts for designing and validating unique directed small molecule toxin transition inhibitors to rapidly prevent toxin transitions under endosomal pH conditions, thus preventing or delaying toxin derived cytotoxic events.